Cell signaling is a part of a complex system of communication that governs basic cellular activities and coordinates cell action. The ability of cells to perceive and correctly respond to their microenvironment is the basis of development, tissue repair and immunity as well as normal tissue homeostasis. Errors in these crucial functions are responsible for diseases such as cancer, autoimmunity and diabetes, and due to the immense role that cell signaling plays, understanding it will provide the cornerstone in understanding diseases and their treatments. Moreover, a cell is capable of sending and receiving chemical signals from other cells, and usually interacts with other cells to achieve coordinated functions rather than exist as a single-cell in its own microenvironment. Therefore, the ability to qualify the cell signaling events is very important for understanding cellular responses.
Modeling of these complex processes will integrate experimental data on the distinct spatial-temporal dynamics of signaling from different cellular compartments and provides new insight into the connection between external stimuli and the signaling outcome in terms of gene expression responses, phenotype response and others. However, large sets of experimental data with the self-consistent and dynamic measures of protein activities are rare in utilizing conventional methods due to the cumbersome and complex nature of the experiments that are required.
One of the possible reasons for these predicaments is related to cellular heterogeneity. In recent studies on the cellular analyses, it has been observed that cells even in genetically identical cell populations under the same environmental conditions exhibit some degrees of variation. Therefore, the information obtained on the cell population level, which is based on the basis of averaged measurements of large group of cells can overlook very important observations or even lead to incorrect results. For this reason, there have been growing interests in single-cell assays to understand single-cell behavior more accurately. Also, cell-cell interactions leading to tissue development involve a small number of cells. So, more pertinent information could be obtained from single-cell level analyses. However, it can be difficult to perform single-cell level analyses using traditional tools because of its labor intensive and low throughput nature, while microfluidic approaches allow more precise control of cell positioning and reagent introduction in analyzing single-cells.
The inventors' earlier work includes the design, fabrication, and testing of a microfluidic chip array containing a plurality of microfluidic chambers each of which comprises a disc-shaped cell chamber that is formed with an initial gap of 5 μm from the underlying substrate to allow a flow stream into and out of the chamber. The chamber can be moved up or down by pneumatic activation using an air chamber that overlies the entire chamber. A cell capture site is formed in the chamber sidewall upstream of the flow and is used to trap an individual cell as the fluid stream moves over the substrate underneath the cell chamber. To load the cell, the air chamber is partially evacuated which, due to the flexibility of the chamber upper wall, enables the 5 μm to be increased large enough to allow the captured cell to move with the flow stream into the space under the now-lifted cell chamber. Thereafter, the air chamber is positively pressurized to push the cell chamber down onto the substrate to thereby completely isolate the cell within the interior region formed by the cell chamber and substrate.
This design, while allowing individual cell capture and assaying, is not designed to permit the introduction into the cell chamber of multiple cells. Thus, it does not permit studying of cell-to-cell interactions which could otherwise be useful in order to elucidate cellular processes such as stem cell proliferation and differentiation. Some recent studies have addressed this problem by allowing observation of single cell pairs in close proximity; however, these studies did not distinguish whether the interaction was induced by secretion or contact from other cells. Also, trapping of the cells within the chamber when it is lifted up to admit the cell can be problematic since the cell can move past the space under the chamber before it is closed or possibly become lodged between the cell chamber sidewall and substrate.